HPV is a pathogen causing approximately 100% of cervical cancer cases [1]. It is reported that, annually, 500,000 women are diagnosed with cervical cancer around the world, and 250,000 women die from cervical cancer [2]. Types 16, 18, 45, 31, 33, 52, 58, 35, and 59 are known as high risk types of HPV that cause cervical cancer, whereas types 6 and 11 are known as low risk types of HPV [3,4]. In particular, HPV16 and HPV18 are known to cause 70% of all cervical cancer cases, and thus are recognized as the most important types in prevention of cervical cancer [5]. Types of HPVs causing cervical cancer vary by region [5]. In Africa, Europe, North America and Central and South America, HPV16, HPV18, HPV31 and HPV45 infections are main causes of cervical cancer, and in Asia, HPV16, HPV18, HPV58 and HPV33 infections are main causes of cervical cancer [5].
A capsid of HPV is composed of L1 protein as a major antigen and L2 protein as a minor antigen [6]. Here, the L1 protein has been used as an antigen for prophylactic vaccine of cervical cancer and an antigen for diagnosis because it has a property of self-assembly that forming virus-like particles (VLPs) [6, 27]. Recombinant L1 protein was produced in Escherichia coli, Saccharomyces cerevisiae, Pichia Pastoris, Lactobacillus casei, or Spodoptera frugiperda (Sf) cells, or plant cells as expression cells [7-12, 28]. Today, commercially available cervical cancer vaccines are Gardasil™ (Merck) and Cervarix™ (GlaxosmithKline, GSK). Gardasil™ includes L1 VLPs with respect to HPV16, HPV18, HPV6 and HPV11 as antigens, and Cervarix™ includes L1 VLPs with respect to HPV16 and HPV18 as antigens [13]. Gardasil™ uses Saccharomyces cerevisiae as an antigen-expressing cell, and Cervarix™ uses Spodoptera frugiperda (Sf) cells, which are insect cells, as antigen-expressing cells [13,14]. Both of the two vaccines are injected by intramuscular injection, and have high costs of $120 for one dose and $360 for three doses [15]. Due to the high injection costs of the commercially available cervical cancer vaccines, the vaccines have many limits to be widely used in a developing country, which is a region in which cervical cancer mainly occurs [16]. Accordingly, development of a low-cost and highly-effective cervical cancer vaccine still remains an important issue.
In the case of a drug using recombinant protein, a cost for downstream processing is as high as 80% of the total production cost [17]. To produce an antigen for the cervical cancer vaccine, a method of sequentially increasing purity of a target antigen through several steps of purification in the downstream processing is used [18]. In the case of VLPs produced in Saccharomyces cerevisiae, to increase the purity of the target protein, sucrose cushion using ultracentrifugation or size-exclusion chromatography has been mainly used. To purify VLPs, Hofmann et al. used a sucrose cushion using ultracentrifugation, anion exchange chromatography, ammonium sulfate precipitation and size-exclusion chromatography [19]. Kim et al. sequentially used a sucrose cushion using ultracentrifugation, size-exclusion chromatography and cation exchange chromatography to purify the VLPs [7]. Park et al. used a purification method including ammonium sulfate precipitation, size-exclusion chromatography, and cation exchange chromatography, which were sequentially performed [20]. According to the methods, high purity VLPs may be obtained, but the methods have a limit in increasing a purification unit, and are not suitable to be applied in large scale production.
For large scale production of VLPs of HPV produced from Saccharomyces cerevisiae, the following methods were used. Cook et al. used a method of sequentially using cross-flow microfiltration, cation exchange chromatography, and hydroxyapatite chromatography [21]. Kim et al. used a method of purifying VLPs through heparin chromatography or cation exchange chromatography after foreign proteins were removed by ammonium sulfate precipitation and step removing precipitated contaminants [22]. A HPV L1 protein produced from Saccharomyces cerevisiae numbers no more than 1% of all known homogenate proteins, and recovery of that is not easy [20]. To purify HPV VLPs produced by a method of preparing a recombinant protein with high purity, several steps of precipitation and chromatography should be performed, and repeated dialysis is also needed. In addition, since the VLPs of HPV are easily disrupted, assembly of the VLPs in an excellent structure is considered to be very important [31]. While researchers have made a lot of effort to develop a process of effectively removing a foreign substance in a homogenate of a host cell such as a yeast expression system, technology for purifying VLPs with high efficiency has not definitely advanced yet.
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